The mitochondrial outer membrane mediates numerous interactions between the mitochondrial genetic and metabolic systems and the rest of the cell, however, details of the biogenesis of this structure and its function on the molecular level remain major unsolved problems of cell biology. The aim of the research described by this proposal is a molecular characterization of a major facet of outer membrane biogenesis: the import of outer membrane proteins. All of these proteins are encoded on nuclear genes, synthesized in the cytoplasm, and directly imported into the outer membrane. This study will employ the yeast Saccharomyces cerevisiae as a model cellular system and will combine a biochemical analysis of the import process with a genetic approach involving mutant yeast strains which are defective in mitochondrial protein import. An initial goal will be the characterization of the import of two major outer membrane proteins, a 45 kd polypeptide and a 15 kd species. The complete genes for these proteins will be isolated and characterized, and subsequently manipulated for use in an import assay involving the synthesis of labeled proteins in an in vitro transcription-translation system programmed with the cloned genes, followed by incubation of the labeled polypeptides with isolated mitochondria or outer membranes. Next, the import of outer membrane proteins will be analyzed in strains previously found to be defective for import of proteins into other sub-mitochondrial compartments. Additional mutant strains, blocked specifically in import of outer membrane proteins, will be isolated and characterized. These studies should provide information concerning the essential steps, energy dependence, involvement of cytosolic factors, and identity of membrane components necessary for import. Mutants specifically defective in the import of outer membrane proteins will then be used to isolate genes for protein components of the import machinery. These can facilitate study of the regulation of outer membrane biogenesis and the relationship of this process to cell growth and differentiation.